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5 Conversion of Proline to Hydroxyproline in Collagen Synthesis

Downloaded by GEORGE MASON UNIV on December 17, 2014 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ba-1964-0044.ch005

ALTON MEISTER, NEVILLE STONE, AND JAMES M. MANNING Department of Biochemistry, Tufts University School of Medicine, Boston, Mass.

Granuloma minces from normal and ascorb a t e - d e f i c i e n t guinea p i g s w e r e incubated with labeled proline; collagen i m i n o acids w e r e subsequently i s o l a t e d and t h e i r s p e c i f i c radioactivities determined. A l t h o u g h the s p e c i f i c a c t i v i t y of p r o l i n e from s c o r b u t i c t i s s u e s w a s not m a r k e d l y r e d u c e d , that of h y d r o x y p r o l i n e w a s m u c h l o w e r than the con trols. Use of proline-Η provided a new pa rameter for following hydroxylation, since H32O formation paralleled that of hydroxyproline-H . There was a marked decrease of H32O formation with ascorbate-deficient gran uloma; addition of ascorbate increased H32O and hydroxyproline-H formation. The data suggest that collagen biosynthesis involves two proline pools; one supplies collagen pro line. The other is oxidized to a hydroxypro line intermediate, not in equilibrium with free hydroxyproline nor in peptide linkage with proline. This hypothesis is considered in relation to currently available information about p r o t e i n s y n t h e s i s . 3

3

3

Collagen i s

the m o s t abundant connective t i s s u e p r o t e i n and c o m p r i s e s as m u c h as 30% of the t o t a l p r o t e i n of s o m e a n i m a l s . A s c o m p a r e d to other p r o t e i n s , c o l l a g e n i s u n u s u a l i n t e r m s of i t s e x t r a c e l l u l a r l o c a t i o n , t e r t i a r y s t r u c t u r e , a m i n o a c i d c o m p o s i t i o n , and a p p a r e n t l y a l s o the m a n n e r of i t s b i o s y n t h e s i s . T h u s , i t a p p e a r s that c o l l a g e n e x i s t s as a t r i p l e - s t r a n d e d h e l i x as f i r s t p r o p o s e d by R a m a c h a n d r a n and K a r t h a (34) and l a t e r e l a b o r a t e d b y o t h e r s (1, 6, 7). A p p r o x i m a t e l y two t h i r d s of the a m i n o a c i d s of c o l l a g e n m a y be accounted f o r by f o u r a m i n o 67

In Amino Acids and Serum Proteins; Stekol, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1964.


68

ADVANCES IN CHEMISTRY SERIES

a c i d s : p r o l i n e , 4 - h y d r o x y p r o l i n e , g l y c i n e , and g l u t a m i c a c i d . C o l l a g e n contains l i t t l e o r none of the s u l f u r - c o n t a i n i n g a m i n o a c i d s , v e r y s m a l l amounts of t y r o s i n e and p h e n y l a l a n i n e , and no tryptophan. C o l l a g e n contains 5 - h y d r o x y l y s i n e , and v e r y r e c e n t l y evidence has been r e p o r t e d of the p r e s e n c e of s m a l l amounts of 3 ^ h y d r o x y p r o l i n e (28). T h e b i o s y n t h e s i s of c o l l a g e n d i f f e r s f r o m that of many o t h e r p r o teins i n s e v e r a l major respects. F o r example, collagen exhibits a v e r y low t u r n o v e r and c e r t a i n c o l l a g e n f r a c t i o n s of a n i m a l s r e m a i n w i t h the a n i m a l f o r i t s l i f e t i m e (17, 49); yet, t h e r e i s evidence of m o r e l a b i l e f o r m s of c o l l a g e n and c e r t a i n of these m a y be p r e c u r s o r s of the m a t u r e i n s o l u b l e c o l l a g e n (17, 18). F r e e h y d r o x y p r o l i n e i s not i n c o r p o r a t e d to an a p p r e c i a b l e extent into c o l l a g e n . T h e e a r l y s t u d i e s of W o m a c k and R o s e (53) showed that h y d r o x y p r o l i n e d i d not r e p l a c e a r g i n i n e i n s u p p o r t i n g the g r o w t h of r a t s , although p r o l i n e and glutamate, u n d e r the s a m e d i e t a r y c o n d i t i o n s , w e r e effective. Stetten and S c h o e n h e i m e r (45, 46), i n e x p e r i m e n t s w i t h N - and d e u t e r i u m - l a b e l e d p r o l i n e , showed that f r e e p r o l i n e r a t h e r than f r e e h y d r o x y p r o l i n e w a s the p r e c u r s o r of c o l l a g e n h y d r o x y p r o l i n e . In this w o r k i t w a s found that h y d r o x y p r o l i n e w a s one twentieth as effective a s p r o l i n e as a p r e c u r s o r of c o l l a g e n h y d r o x y p r o l i n e . T h e s e s t u d i e s , w h i c h have been c o n f i r m e d by o t h e r s (15, 26), have l e d to the suggestion (45) that h y d r o x y l a t i o n of p r o l i n e takes p l a c e i n peptide l i n k a g e . S i m i l a r c o n c l u s i o n s m a y apply to the 5h y d r o x y l y s i n e of c o l l a g e n ; thus, l y s i n e r a t h e r than h y d r o x y l y s i n e i s the p r e c u r s o r of c o l l a g e n h y d r o x y l y s i n e (31, 42). C o l l a g e n s y n t h e s i s takes p l a c e at a r e d u c e d r a t e i n a s c o r b i c a c i d d e f i c i e n c y (14, 38, 39, 52). F o r e x a m p l e , g r a n u l a t i o n t i s s u e induced by wounding, o r by i n t r o d u c t i o n of a f o r e i g n substance s u c h as p o l y s a c c h a r i d e o r a p o l y v i n y l sponge, contains m u c h l e s s c o l l a g e n (and h y d r o x y p r o l i n e ) when s u c h p r o c e d u r e s a r e c a r r i e d out i n a s c o r b i c a c i d deficient guinea p i g s . T h e function of a s c o r b i c a c i d i n c o l l a g e n s y n t h e s i s i s not yet known. It has been suggested that i t i s r e q u i r e d f o r the maintenance of c o l l a g e n (16), f o r h y d r o x y l a t i o n of peptide-bound p r o l i n e , and f o r the m a t u r a t i o n of f i b r o b l a s t s (25); i t m i g h t a l s o function i n a h o r m o n a l l y induced s y s t e m i c effect. Studies by G o u l d (12) and R o b e r t son and H e w i t t (37), and i n t h i s l a b o r a t o r y (47, 48) suggest that t h i s v i t a m i n has a d i r e c t function i n h y d r o x y l a t i o n . T h e p r e s e n t w o r k w a s u n d e r t a k e n i n an attempt to shed l i g h t on the m e c h a n i s m of h y d r o x y p r o l i n e f o r m a t i o n i n c o l l a g e n b i o s y n t h e s i s and on the s i t e of a c t i o n of a s c o r b a t e . It w a s r e c o g n i z e d that these phenomena w e r e v e r y l i k e l y to be c l o s e l y i n t e r r e l a t e d . P r o l i n e l a b e l e d w i t h t r i t i u m was u s e d because i t was anticipated that t r i t i u m r e l e a s e d f r o m p r o l i n e d u r i n g h y d r o x y l a t i o n w o u l d appear i n the t i s s u e w a t e r a s t r i t i a t e d w a t e r . T h e use of t r i t i a t e d p r o l i n e i n s u c h a s y s t e m m i g h t then p r o v i d e a new p a r a m e t e r f o r f o l l o w i n g the h y d r o x y l a t i o n r e a c t i o n , p r o v i d e d that the f o r m a t i o n of t r i t i a t e d w a t e r w a s s t o i c h i o m e t r i c a l l y r e l a t e d to the f o r m a t i o n of h y d r o x y p r o l i n e . Such an a p p r o a c h m i g h t c o n c e i v a b l y p e r m i t the d e m o n s t r a t i o n of h y d r o x y l a t i o n and peptide bond f o r m a t i o n as s e p arate c h e m i c a l steps. M i n c e s of c o l l a g e n - f o r m i n g g r a n u l o m a s w e r e incubated w i t h m e d i a 1

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Conversion of Proline to Hydroxyproline

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1 4


c o n t a i n i n g C - l a b e l e d o r t r i t i a t e d p r o l i n e (or other l a b e l e d a m i n o a c i d s ) . T h e t u m o r s w e r e i n d u c e d i n guinea p i g s by i n j e c t i o n of c a r r a g e e n i n as d e s c r i b e d b y R o b e r t s o n and S c h w a r t z (39). G u i n e a p i g s fed n o r m a l d i e t s and a s c o r b a t e - d e f i c i e n t d i e t s w e r e e m p l o y e d . " G e n e r a l l y labeled** t r i t i a t e d p r o l i n e and t r i t i a t e d p r o l i n e obtained b y t r i t i a t i o n of 3 , 4 - d e h y d r o p r o l i n e (40) w e r e u s e d . T h e t r i t i a t e d p r o l i n e p r e p a r a t i o n s w e r e obtained f r o m the N e w E n g l a n d N u c l e a r C o r p . , S c h w a r z B i o r e s e a r c h , I n c . , and C o m m i s s a r i a t l ' É n e r g i e A t o m i q u e , G i f - s u r Yvette, France. C o l l a g e n and the noncollagenous p r o t e i n s w e r e i s o l a t e d by the g e n e r a l p r o c e d u r e i n d i c a t e d i n F i g u r e 1. T h e t i s s u e w a s homogenized i n 1. Tissue homogenized in 75% acetone 2. Homogenate centrifuged (1000 x g) 3. Residue washed in 95% ethanol (6x); ether-ethanol (3:1) (2x; 60°; 10 minutes); 95% ethanol 4. Collagen extracted from residue with 5% trichloroacetic acid (2x; 90°)

5. Extracts centrifuged at 10,000 x g; collagen precipitated by addition of tannic acid at 0° 6. Collagen-tannate washed with 5% tannic acid at 0°

Figure

7. Residue washed with 5% trichloroacetic acid and 95% ethanol 8. Residue dissolved in Ν NaOH; 60°; 10 minutes; reprecipitated by addition of HCl and trichloro acetic acid 9. Residue (noncollagenous protein) washed with 95% ethanol

1. Scheme for Extraction

of

Collagen

acetone, t r e a t e d w i t h other s o l v e n t s , and then e x t r a c t e d w i t h hot t r i c h l o r o a c e t i c a c i d . F i n a l l y , the c o l l a g e n w a s p r e c i p i t a t e d b y a d d i t i o n of tannic a c i d . T h e i m i n o a c i d s w e r e obtained f r o m a c i d h y d r o l y z a t e s of the c o l l a g e n by i o n - e x c h a n g e c h r o m a t o g r a p h y after d e s t r u c t i o n of the α - a m i n o a c i d s by t r e a t m e n t w i t h n i t r o u s a c i d (38). In s o m e e x p e r i ments the i m i n o a c i d s w e r e obtained b y p a p e r c h r o m a t o g r a p h y and electrophoresis. T h e c o l l a g e n i s o l a t e d f r o m d e f i c i e n t and c o n t r o l g r a n u l o m a s e x h i b i t e d a p p r o x i m a t e l y the s a m e a m i n o a c i d c o m p o s i t i o n as that of p u r i f i e d c o m m e r c i a l g e l a t i n . The a m i n o a c i d c o m p o s i t i o n w a s e x a m i n e d both by t w o - d i m e n s i o n a l paper c h r o m a t o g r a p h y and by g a s - l i q u i d c h r o m a t o g raphy (19). The r a t i o of p r o l i n e to h y d r o x y p r o l i n e was v i r t u a l l y i d e n t i c a l f o r c o l l a g e n e x t r a c t e d f r o m n o r m a l and deficient g r a n u l o m a s ( T a b l e I). F u r t h e r i n d i c a t i o n of the p u r i t y of the i s o l a t e d c o l l a g e n i s the fact that c o l l a g e n does not contain tryptophan. T h u s , when ^ - t r y p t o phan w a s incubated w i t h m i n c e s of the g r a n u l o m a , s i g n i f i c a n t quantities of r a d i o a c t i v i t y w e r e i n c o r p o r a t e d into the noncollagenous p r o t e i n but



70

Table I. Mole Ratio of Proline to Hydroxyproline in Collagen Isolated from Granuloma of Normal and


Scorbutic Guinea Pigs

0

Source of Granuloma

Mole Ratio Proline/Hydroxyproline

Normal Normal Scorbutic Scorbutic

1.24 1.28 1.24 1.26

a

Collagen isolated from granuloma as tannate and hydrolyzed in 6N HCl; imino acids isolated as described in text, and quantitatively determined (27, 59).

not into the c o l l a g e n (Table Π ) . W a t e r w a s obtained f r o m the r e a c t i o n m i x t u r e s by l y o p h i l i z a t i o n . T h e r a d i o a c t i v i t y of the i s o l a t e d i m i n o a c i d s and the t r a p p e d w a t e r w a s d e t e r m i n e d by counting w i t h a C h i c a g o N u c l e a r C o . d u a l - w i n d o w s c i n t i l l a t i o n c o u n t e r . In s o m e of the e x p e r i m e n t s w i t h C - a m i n o a c i d s , an a u t o m a t i c gas flow m i c a window counter was employed. In the e x p e r i m e n t s r e p o r t e d i n T a b l e ΙΠ 2 g r a m s of g r a n u l o m a m i n c e w e r e incubated w i t h 5 m l . of m e d i u m containing 5 μ ο . of " 3 , 4 t r i t i a t e d (batch 64-121-1) L - p r o l i n e (173 μ ο . p e r μ ι η ο ΐ β ) o r 1 μ ο . of DL-proline-l-C (3.1 μ ο . p e r μ π ι ο ΐ β ) (obtained f r o m the N e w E n g l a n d N u c l e a r C o r p . , B o s t o n , M a s s . ) . Incubation was c a r r i e d out at 3 7 ° C . under 95% o x y g e n - 5 % c a r b o n d i o x i d e . T h e m e d i u m contained 0 . 0 2 2 M N a C l , 0.003M K C 1 , 0.0012M M g S 0 , 0.0013M C a C ^ , 0.0004M K H P 0 , 0 . 0 2 5 M N a H C 0 , and 0 . 0 1 M D - g l u c o s e . T r i t i u m and c a r b o n - 1 4 w e r e d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n counting. T h e deficient a n i m a l s w e r e d e p r i v e d of a s c o r b i c a c i d f o r 2 w e e k s . P r o l i n e and h y d r o x y p r o l i n e w e r e 1 4

, ,

1 4

4

2

3

14

Table II. Incorporation of C -Tryptophan into Collagen and Noncollagenous Proteins of Guinea Pig Granuloma

Expt. No.

Noncollagenous Protein, C.p.m./Mg.

Collagen Tannate, C.p.m./Mg. (Approx.)

1 2 3 4 5 6

108 94 17 40 27 69

2 3 1 1 0 1

a

4

14

Reaction mixtures contained 7 χ 10 c.p.m. of 3 - C - D L tryptophan(10 curies/mole); incubated for 4 hours; other conditions as in Table ΙΠ. Noncollagenous proteins proc essed by method of Siekevitz (41).


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Table III. Incorporation of Proline into Collagen Proline and Hydroxyproline of Granulomas from Normal and Scorbutic Guinea Pigs

Specific Activity, D.P.M./fcmole

Isotope


H

Expt. No.

3

Incuba tion Period, Min. 30 60 120 30 30

Normal

Deficient

Proline

Hydroxyproline

Proline

Hydroxyproline

6,150 13,300 21,300 790 745

2,400 5,070 8,700 695 680

4,580 9,300 17,800 787 530

< 100 150 360 < 10 70

i s o l a t e d f r o m a c i d h y d r o l y z a t e s of the c o l l a g e n by p a p e r c h r o m a t o g raphy (solvent 1 - b u t a n o l - a c e t i c a c i d - w a t e r , 4:1:1) and d e t e r m i n e d , r e s p e c t i v e l y , as d e s c r i b e d by T r o l l and L i n n s l e y (50) a n d N e u m a n and L o g a n (27). Results A s i n d i c a t e d i n F i g u r e 2, when m i n c e s of t u m o r obtained f r o m n o r m a l and a s c o r b i c a c i d - d e f i c i e n t a n i m a l s w e r e incubated w i t h C - p r o l i n e , m u c h m o r e r a d i o a c t i v i t y w a s i n c o r p o r a t e d into the c o l l a g e n of the n o r m a l t i s s u e . When the s p e c i f i c r a d i o a c t i v i t i e s of the i s o l a t e d i m i n o a c i d s w e r e e x a m i n e d (Table Π Ι ) , s e v e r a l c o n c l u s i o n s w e r e p o s s i b l e . I n the e x p e r i m e n t s w i t h g r a n u l o m a f r o m n o r m a l a n i m a l s and C - p r o l i n e , the v a l u e s f o r h y d r o x y p r o l i n e w e r e not f a r f r o m those of p r o l i n e . W i t h the s c o r b u t i c g r a n u l o m a s , the s p e c i f i c a c t i v i t y of the i s o l a t e d p r o l i n e w a s not g r e a t l y r e d u c e d , c o m p a r e d to that obtained f r o m the n o r m a l g r a n u l o m a . I n c o n t r a s t , the s p e c i f i c a c t i v i t y of the h y d r o x y p r o l i n e i s o lated f r o m the deficient t i s s u e s w a s m a r k e d l y r e d u c e d . S i m i l a r r e s u l t s w e r e obtained i n the studies w i t h t r i t i a t e d p r o l i n e . T h u s , the s p e c i f i c a c t i v i t y of the p r o l i n e i s o l a t e d f r o m the deficient g r a n u l o m a w a s only m o d e r a t e l y r e d u c e d , w h e r e a s the s p e c i f i c a c t i v i t y of the h y d r o x y p r o l i n e w a s e x t r e m e l y l o w . T h i s o b s e r v a t i o n m a y be explained i n t e r m s of a dual-pathway m e c h a n i s m of p r o l i n e i n c o r p o r a t i o n , c o n s i d e r e d below. In c o n t r a s t to the r e s u l t s obtained w i t h C - p r o l i n e , the h y d r o x y p r o l i n e i s o l a t e d f r o m the n o r m a l g r a n u l o m a i n the e x p e r i m e n t s w i t h t r i t i a t e d p r o l i n e exhibited a m u c h l o w e r s p e c i f i c a c t i v i t y than the p r o l i n e i s o l a t e d f r o m the s a m e s o u r c e . T h e s e findings i n d i c a t e that t r i t i u m w a s l o s t f r o m p r o l i n e d u r i n g h y d r o x y l a t i o n ; d e t e r m i n a t i o n of the t r i t i u m - C r a t i o s f o r p r o l i n e and h y d r o x y p r o l i n e obtained f r o m n o r m a l c o l l a g e n (Table I V ) i n d i c a t e s that a p p r o x i m a t e l y one half of the p r o l i n e t r i t i u m w a s l o s t d u r i n g h y d r o x y l a t i o n . R e s u l t s consistent w i t h t h i s c o n c l u s i o n a r e shown i n T a b l e V , w h i c h g i v e s the t o t a l v a l u e s f o r the r a d i o a c t i v i t i e s of the w a t e r and the c o l l a g e n h y d r o x y p r o l i n e . T h u s , t h e r e w a s , w i t h i n e x p e r i m e n t a l e r r o r , a s m u c h isotope found i n the w a t e r a s i n the c o l l a g e n h y d r o x y p r o l i n e . F i g u r e 3 shows the d r a m a t i c effect of a s c o r b i c a c i d d e p r i v a t i o n on 1 4

1 4

1 4

1 4



72


ASCORBIC ACIDDEFICIENT

MINUTES Figure

120

2. In vitro incorporation of C into granuloma collagen Experimental

details

lA

-proline

as in Table III

t r i t i a t e d w a t e r f o r m a t i o n . T h e a v a i l a b l e data suggest that v i r t u a l l y a l l of the t r i t i a t e d w a t e r f o r m e d i s a s s o c i a t e d w i t h h y d r o x y l a t i o n of p r o l i n e . In studies w i t h both C - p r o l i n e and t r i t i a t e d p r o l i n e we have not 1 4

Table IV. Ratios of Tritium to Carbon-14 for Proline and Hydroxyproline Isolated fron Collagen 3

1 4

Ratio

Incubation Time, Minutes

Hydroxyproline

Β Proline

Ratio A / B

1

30 60 120

3.45 2.65 2.72

7.80 4.53 4.78

0.44 0.58 0.57

2

30 60 120

2.98 2.60 2.60

4.80 4.73 4.62

0.62 0.55 0.56

Expt. No.

l

H /C

0

14

3

C -proline and H -proline used together; experimental condi tions as in Table ΙΠ.


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Table V. Radioactivities of Tritiated Hydroxyproline and Tritiated Water after Incorporation of Tritiated Proline into Collagen

Expt. No.


1 2 3

State of Animal

Hydroxyproline, D.P.M.

HfO, D.P.M.

62,000 46,300 11,500 22,100 20,500

61,000 53,000 13,900 20,400 27,100

Normal Normal Deficient + 50 μ g . / m l . ascorbate + 80 Mg./ml. ascorbate

Experimental conditions as in Table III.

found the isotope i n glutamate o r other a m i n o a c i d s . S i m i l a r o b s e r v a tions w e r e made by G r e e n and L o w t h e r (15) w i t h C - p r o l i n e . It t h e r e f o r e a p p e a r s that i n t h i s t u m o r m e t a b o l i s m of p r o l i n e to p r o d u c t s other than h y d r o x y p r o l i n e i s n e g l i g i b l e . T h e v e r y low l e v e l of t r i t i a t e d w a t e r f o r m a t i o n i n the deficient t i s s u e s ( F i g u r e 3) i s a l s o consistent w i t h the absence of s i g n i f i c a n t a l t e r n a t i v e pathways of p r o l i n e m e t a b o l i s m under these c o n d i t i o n s . T h e data g i v e n i n T a b l e V I show that t h e r e w a s a c o n s i d e r a b l e i n c r e a s e i n f o r m a t i o n of t r i t i a t e d w a t e r upon i n v i t r o addition of a s c o r b i c a c i d . A d d i t i o n of a s c o r b i c a c i d f a i l e d to i n c r e a s e t r i t i a t e d w a t e r f o r m a t i o n w i t h m i n c e s of n o r m a l g r a n u l o m a , and no effect w a s o b s e r v e d 1 4

Conditions

as in Table III



74

Table VI. Stimulation of Hydroxylation of Proline *by Ascorbic Acid


Deficient Granuloma by in Vitro Addition of Ascorbic Acid

Expt. No.

Time

1

Deficient

2

Deficient

3

Normal

4

Normal b

Normal (boiled) ^

b

Ascorbate Added, Mg./Ml.

Tritiated Water, D.P.M.

None 800 None 20 100 None 20 100 None 100 800 None 100 800

19,700 45,500 14,400 25,700 23,100 27,200 30,600 26,500 7,850 8,050 8,550 880 790 790

Experimental conditions as in Table ΠΙ. l g r a m of mince and 1.3 x 10 d.p.m. of tritiated proline used; final volume, 2.5 ml. 6

w i t h p r e p a r a t i o n s that had been i n a c t i v a t e d b y b o i l i n g f o r 5 minutes p r i o r to the e x p e r i m e n t . That a d d i t i o n of a s c o r b i c a c i d i s a s s o c i a t e d a l s o w i t h an i n c r e a s e of h y d r o x y p r o l i n e f o r m a t i o n i s shown i n T a b l e V ; the r e l a t i v e amounts of t r i t i a t e d w a t e r and t r i t i a t e d h y d r o x y p r o l i n e r e m a i n the s a m e under these c o n d i t i o n s . W h i l e these studies w e r e i n p r o g r e s s , R o b e r t s o n and H e w i t t (37) r e p o r t e d that i n v i t r o a d d i t i o n of a s c o r b i c a c i d to b r e i s of a s c o r b i c a c i d - d e f i c i e n t g r a n u l a t i o n t i s s u e s l e d to a n i n c r e a s e i n the i n c o r p o r a t i o n of C - p r o l i n e into c o l l a g e n h y d r o x y p r o l i n e . T h e s e r e s u l t s s t r o n g l y suggest that a s c o r b i c a c i d e x e r t s a d i r e c t a c t i o n on h y d r o x y l a t i o n and r e n d e r l e s s l i k e l y the p o s s i b i l i t y that the effect of a s c o r b a t e i s h o r m o n a l l y mediated o r that i t i s r e q u i r e d f o r the m a t u r a t i o n of f i b r o b l a s t s . T h e studies w i t h t r i t i a t e d p r o l i n e have p r o v i d e d data w h i c h appear to be of s i g n i f i c a n c e i n r e l a t i o n to the o v e r - a l l m e c h a n i s m of c o l l a g e n s y n t h e s i s and the effect of a s c o r b a t e . It s e e m e d p o s s i b l e , at l e a s t i n i t i a l l y , that these e x p e r i m e n t s m i g h t a l s o elucidate c e r t a i n a s p e c t s of the c h e m i c a l m e c h a n i s m of h y d r o x y l a t i o n . T h u s , a s i n d i c a t e d i n T a b l e s I V and V , a p p r o x i m a t e l y one half of the t r i t i u m of the t r i t i a t e d p r o l i n e u s e d i n these e x p e r i m e n t s w a s l o s t d u r i n g h y d r o x y l a t i o n . A s stated p r e v i o u s l y (48), " t h e s e findings a r e c o n s i s t e n t w i t h h y d r o x y l a t i o n m e c h a n i s m s i n v o l v i n g e i t h e r a 4-keto i n t e r m e d i a t e o r 3 , 4 - u n s a t u r a t i o n , a s s u m i n g r a n d o m d i s t r i b u t i o n of t r i t i u m at p r o l i n e c a r b o n s 3 a n d 4, and absence of s i g n i f i c a n t isotope e f f e c t s . " E b e r t and P r o c k o p (8) i n c o r r e c t l y stated that Stone and M e i s t e r " o b s e r v e d a l o s s of both hydrogens f r o m the c a r b o n p o s i t i o n on w h i c h h y d r o x y l a t i o n o c c u r s . " R e c e n t s t u d i e s (9, 10, 33) i n d i c a t e that the 4 - o x y g e n a t o m of h y d r o x y p r o l i n e i n 1 4



5.

MEISTER ET 4 L


75

c h i c k e m b r y o s a r i s e s f r o m a t m o s p h e r i c oxygen r a t h e r than w a t e r . T h e s e findings suggest that an oxygenase m e c h a n i s m i s i n v o l v e d i n the h y d r o x y l a t i o n of p r o l i n e , and r e n d e r u n l i k e l y the f o r m a t i o n of a 3 , 4 - u n s a t u r a t e d i n t e r m e d i a t e w h i c h subsequently adds w a t e r . S m i t h and M i t o m a (43) have found a n e n z y m e a c t i v i t y i n m a m m a l i a n t i s s u e s capable of r e d u c i n g 4 - k e t o - L - p r o l i n e to L - h y d r o x y p r o l i n e ; s u c h a r e a c t i o n ( p e r h a p s i n v o l v i n g bound f o r m s of k e t o p r o l i n e o r h y d r o x y p r o l i n e ) might c o n c e i v a b l y function i n h y d r o x y p r o l i n e f o r m a t i o n . In an attempt to l e a r n whether c o l l a g e n h y d r o x y p r o l i n e s y n t h e s i z e d i n g r a n u l o m a m i n c e s incubated w i t h t r i t i a t e d p r o l i n e contained t r i t i u m at the 4 p o s i t i o n , we converted s u c h t r i t i a t e d h y d r o x y p r o l i n e to the c o r r e s p o n d i n g N - c a r b o b e n z o x y d e r i v a t i v e and then o x i d i z e d t h i s d e r i v a t i v e w i t h c h r o m i c a c i d to N - c a r b o b e n z o x y - 4 - k e t o p r o l i n e a c c o r d i n g to the p r o c e d u r e of Patchett and W i t k o p ( 2 9 ) . I n m o s t of these e x p e r i m e n t s v e r y l i t t l e t r i t i a t e d w a t e r w a s f o r m e d ; although s o m e t r i t i a t e d w a t e r was detected i n s e v e r a l e x p e r i m e n t s , the s m a l l amount f o r m e d could l o g i c a l l y be attributed to adventitious o x i d a t i v e r e a c t i o n s o r p e r h a p s to e n o l i z a t i o n of k e t o p r o l i n e . T h e evidence w a s therefore consistent w i t h the absence of t r i t i u m at c a r b o n a t o m 4 of the i s o l a t e d h y d r o x y p r o l i n e . A n y c o n c l u s i o n s d r a w n f r o m these e x p e r i m e n t s r e s t upon t h r e e a s s u m p t i o n s : that the t r i t i a t e d p r o l i n e w a s l a b e l e d i n the 3 and 4 p o s i tions o n l y , that the l a b e l w a s r a n d o m l y d i s t r i b u t e d i n these p o s i t i o n s , and that no s i g n i f i c a n t isotope effects o c c u r r e d . Studies analogous to those d e s c r i b e d i n T a b l e V w e r e c a r r i e d out w i t h two p r e p a r a t i o n s of " g e n e r a l l y l a b e l e d " t r i t i a t e d p r o l i n e s . T h e r e s u l t s (Table VII) i n d i c a t e that two sevenths of the p r o l i n e t r i t i u m w a s Table VII. Radioactivities of Tritiated Water and Tritiated Hydroxyproline after Incorporation of Generally Tritiated Proline into Collagen

Expt. No.

Tritiated Water, D.P.M. (A)

Hydroxyproline, D.P.M. (B)

Ratio A / A + Β

a

8,350 8,800 139,000

20,100 21,200 334,000

0.29 0.29 0.29

l 2 3b a

a

2 grams of minced granuloma (from normal animal) and 5.0 μο. of generally labeled tritiated DL-proline (Comissariat Γ énergie Atomique; 44 curies/mole) used; incubation period, 2 hours. ^15μο. of generally labeled tritiated L-proline (Schwarz BioResearch, Inc.; 1000 curies/mole) used. Other conditions as in Table ΠΙ.

l i b e r a t e d as t r i t i a t e d w a t e r d u r i n g h y d r o x y l a t i o n . A l t h o u g h the r e s u l t s w i t h the two different p r e p a r a t i o n s of t r i t i a t e d p r o l i n e w e r e the s a m e , c o n c l u s i o n s c o n c e r n i n g the s p e c i f i c p o s i t i o n s f r o m w h i c h t r i t i u m i s l o s t r e q u i r e a s s u m p t i o n s s i m i l a r to those g i v e n above. T h e " g e n e r a l l y l a b e l e d " p r o l i n e p r e p a r a t i o n used i n e x p e r i m e n t s 1 and 2 w a s c o n v e r t e d to p y r r o l i d - 2 - o n e h y d r o c h l o r i d e a s d e s c r i b e d b y B r a g g and Hough (2). T h e s p e c i f i c a c t i v i t y of the p r o l i n e w a s 5.7 x 1 0 8



76

c . p . m . p e r m o l e , and that of the p r o d u c t [ ( p y r r o l i d - 2 - o n e ) H C l ] was 1.06 x 10 c . p . m . p e r m o l e . T h e r a t i o of these v a l u e s i s 0.54; a r a t i o of 0.58 w o u l d be expected f o r u n i f o r m l y t r i t i a t e d p r o l i n e . That one o r m o r e of these a s s u m p t i o n s m a y be open to question i s suggested by the v e r y r e c e n t s t u d i e s of E b e r t and P r o c k o p (8) and by subsequent w o r k i n our l a b o r a t o r y w i t h another b a t c h (No. 73-253) of " 3 , 4 - t r i t i a t e d " p r o l i n e obtained f r o m the New E n g l a n d N u c l e a r C o r p . In e x p e r i m e n t s w i t h t h i s m a t e r i a l , l e s s t r i t i u m a p p e a r e d i n the w a t e r than i n the studies d e s c r i b e d i n T a b l e V . T h u s , under the conditions of the studies d e s c r i b e d i n T a b l e V , 96,800 d . p . m . of t r i t i a t e d w a t e r and 186,000 d . p . m . of t r i t i a t e d h y d r o x y p r o l i n e w e r e found. In e x p e r i m e n t s w i t h both " u n i f o r m l y l a b e l e d " C - p r o l i n e and the new batch of " 3 , 4 tritiated" proline, H / C r a t i o s f o r h y d r o x y p r o l i n e and p r o l i n e w e r e 1.79 and 2.56, r e s p e c t i v e l y , l e a d i n g to an A / B v a l u e (cf. T a b l e I V ) of 0.70. E b e r t and P r o c k o p (8) c a r r i e d out s i m i l a r s t u d i e s w i t h " 3 , 4 - t r i t i a t e d " p r o l i n e i n c h i c k e m b r y o s , and r e p o r t e d r a t i o s of 0.73 and 0.74. I n f o r m a t i o n s u p p l i e d by P r o c k o p (32) i n d i c a t e s that this m a t e r i a l was f r o m a batch (New E n g l a n d N u c l e a r C o r p . ) different f r o m those used by u s . T h e y c o n v e r t e d the i s o l a t e d h y d r o x y p r o l i n e to p y r r o l e and found r a t i o s of the H / C v a l u e s f o r p y r r o l e to h y d r o x y p r o l i n e of 0.61 and 0.64 (these v a l u e s w e r e c o r r e c t e d for l o s s of c a r b o x y l C , a s s u m i n g r a n d o m l a b e l i n g ) . W e have r e c e n t l y c o n v e r t e d the H - h y d r o x y p r o l i n e obtained i n our e x p e r i m e n t s w i t h the new batch of " 3 , 4 - t r i t i a t e d " p r o l i n e to p y r r o l e (32), and obtained v a l u e s of 0.70 and 0.73. H o w e v e r , c o n v e r s i o n of t h i s h y d r o x y p r o l i n e to Ν - c a r b o b e n z o x y - 4 - k e t o p r o l i n e d i d not lead to a s i g n i f i c a n t d e c r e a s e i n s p e c i f i c a c t i v i t y , s u g g e s t i n g that l i t t l e t r i t i u m w a s p r e s e n t at c a r b o n a t o m 4 of the i s o l a t e d h y d r o x y p r o line. It i s evident that the studies w i t h t r i t i a t e d p r o l i n e do not p e r m i t definite c o n c l u s i o n s about the m e c h a n i s m of h y d r o x y l a t i o n ; f u r t h e r i n f o r m a t i o n i s needed about the l a b e l i n g of the p r o l i n e used and p o s s i b l e isotope effects. It i s p r o b a b l e that t h e r e i s s o m e r a n d o m i z a t i o n of l a b e l d u r i n g t r i t i a t i o n of 3 , 4 - d e h y d r o p r o l i n e (40), and apparently a l s o s o m e r a c e m i z a t i o n . T h u s , we have o b s e r v e d that the t r i t i a t e d p r o l i n e p r e p a r a t i o n s contain 10 to 20% D - p r o l i n e and that o x i d a t i o n by D - a m i n o a c i d o x i d a s e r e l e a s e s s i g n i f i c a n t amounts of t r i t i a t e d w a t e r . D e s p i t e the d i v e r g e n t r e s u l t s obtained w i t h different p r e p a r a t i o n s of t r i t i a t e d p r o l i n e , we have found that the r a t i o of the t r i t i a t e d w a t e r f o r m e d to the t r i t i a t e d h y d r o x y p r o l i n e f o r m e d i s constant f o r a given batch of t r i t i a t e d p r o l i n e . Since i n the c a r r a g e e n i n t u m o r s y s t e m t h e r e i s a definite r e l a t i o n s h i p between the f o r m a t i o n of t r i t i a t e d w a t e r and t r i t i a t e d h y d r o x y p r o l i n e , t h i s s y s t e m should be of v a l u e i n obtaining d e f i n i t i v e r e s u l t s when a p p r o p r i a t e l y l a b e l e d p r o l i n e s a r e a v a i l a b l e . T h i s i s apparently not t r u e f o r the c h i c k e m b r y o , i n w h i c h t h e r e i s a c o n s i d e r a b l e m e t a b o l i s m of p r o l i n e to other p r o d u c t s and t h e r e f o r e a m u c h g r e a t e r f o r m a t i o n of t r i t i a t e d w a t e r (23). In the o r i g i n a l studies of Stetten and Schoenheimer (46) (who e m ployed p r o l i n e l a b e l e d w i t h N and d e u t e r i u m i n p o s i t i o n s 3, 4, and 5), N : D r a t i o s of 0.18 and 0.13 w e r e obtained f o r the i s o l a t e d h y d r o x y 2


9

1 4

3

3

1 4

1 4

1 4

3

1 5

1 5



5.

MEISTER ET AL.


77

p r o l i n e and p r o l i n e , r e s p e c t i v e l y . The r a t i o of the v a l u e obtained f o r p r o l i n e to that for h y d r o x y p r o l i n e was t h e r e f o r e 0.72. A v a l u e of 0.83 would have been expected f o r the l o s s of one a t o m of d e u t e r i u m , w h i l e 0.67 m a y be c a l c u l a t e d f o r the l o s s of two a t o m s of d e u t e r i u m . The r e s u l t of Stetten and S c h o e n h e i m e r t h e r e f o r e l i e s between these two c a l culated v a l u e s , although c l o s e r to a v a l u e consistent w i t h the l o s s of two a t o m s of d e u t e r i u m . A l t h o u g h we m u s t tentatively put a s i d e the question of the d e t a i l e d c h e m i c a l m e c h a n i s m of h y d r o x y l a t i o n , the p r e s e n t data appear to p r o v i d e i n s i g h t into the r e l a t i o n s h i p between h y d r o x y l a t i o n and c o l l a g e n s y n t h e s i s . T h u s , we may f i r s t c o n s i d e r a m e c h a n i s m as shown below, based on the suggestion of Stetten. P r o l i n e — « - [ ( p r o ) -peptide]

1

n

[ (pro) _x-(hypro)x-peptide] n

collagen--

1

A c c o r d i n g to s u c h a s c h e m e , p r o l i n e i s i n c o r p o r a t e d into c o l l a g e n through a s e r i e s of p r e c u r s o r peptides; u l t i m a t e l y about half of the p r o l i n e r e s i d u e s of s u c h peptides a r e c o n v e r t e d to h y d r o x y p r o l i n e . If this m e c h a n i s m w e r e c o r r e c t , one w o u l d expect a p r o l i n e - r i c h c o l l a g e n p r e c u r s o r to a c c u m u l a t e i n s c u r v y . H o w e v e r , s a t i s f a c t o r y evidence f o r this type of i n t e r m e d i a t e has not yet been p u b l i s h e d . In a d d i t i o n , our data i n d i c a t e i d e n t i c a l p r o l i n e - h y d r o x y p r o l i n e r a t i o s f o r c o l l a g e n o b tained f r o m s c o r b u t i c and n o r m a l g r a n u l o m a s . A c c o r d i n g to t h i s s c h e m e , the r a t i o of the s p e c i f i c a c t i v i t y of c o l l a g e n h y d r o x y p r o l i n e to that of c o l l a g e n p r o l i n e should not exceed unity. H o w e v e r , r a t i o s m u c h g r e a t e r than unity have been found by G r e e n and L o w t h e r (15), who have o b s e r v e d r a t i o s a s high as 1.69. W e have made s i m i l a r o b s e r v a t i o n s . T h e s t r i k i n g f a l l i n the s p e c i f i c a c t i v i t y of the c o l l a g e n h y d r o x y p r o l i n e i s o l a t e d f r o m deficient g r a n u l o m a s o b s e r v e d i n the p r e s e n t study (Table ΠΙ) suggests an a l t e r n a t i v e s c h e m e of c o l l a g e n s y n t h e s i s .

A c c o r d i n g to t h i s p r o p o s a l , p r o l i n e i s c o n v e r t e d to c o l l a g e n p r o l i n e and c o l l a g e n h y d r o x y p r o l i n e by two s e p a r a t e and d i s t i n c t pathways. P r o l i n e destined to become h y d r o x y p r o l i n e i s o x i d i z e d to a bound h y d r o x y p r o l i n e i n t e r m e d i a t e , w h i c h does not e q u i l i b r a t e s i g n i f i c a n t l y w i t h f r e e h y d r o x y p r o l i n e and i s not yet i n peptide linkage w i t h p r o l i n e . The m a r k e d d r o p i n the s p e c i f i c a c t i v i t y of h y d r o x y p r o l i n e i n s c o r b u t i c c o l lagen m a y then be e x p l a i n e d by the a c c u m u l a t i o n of a n i n t e r m e d i a t e o r i n t e r m e d i a t e s i n the h y d r o x y p r o l i n e pathway. Such a c c u m u l a t i o n w o u l d




78

l e a d to a m u c h g r e a t e r d i l u t i o n of the p r o l i n e destined to become c o l l a gen h y d r o x y p r o l i n e than p r o l i n e w h i c h w i l l become c o l l a g e n p r o l i n e . A b l o c k at e i t h e r step 2 o r step 3 i n the s c h e m e g i v e n h e r e w o u l d be e x pected to lead to the o b s e r v e d d e c r e a s e i n f o r m a t i o n of t r i t i a t e d w a t e r . It a p p e a r s u n l i k e l y that i n s c u r v y t h e r e i s a b l o c k between f r e e p r o l i n e and the f i r s t bound p r o l i n e i n t e r m e d i a t e . Such a b l o c k w o u l d l e a d to p r o l i n e a c c u m u l a t i o n and thus d i l u t e p r o l i n e for both pathways. S i m i l a r c o n s i d e r a t i o n s i n d i c a t e that i n t e r m e d i a t e I and i n t e r m e d i a t e Π cannot be i d e n t i c a l . It i s c o n c e i v a b l e that the r e d u c e d f o r m a t i o n of t r i t i a t e d w a t e r o b s e r v e d i n s c u r v y c o u l d be due to a g e n e r a l r e d u c t i o n i n p r o t e i n synthe s i s , but t h i s w o u l d not e x p l a i n the m a r k e d differences between the s p e c i f i c a c t i v i t i e s of p r o l i n e and h y d r o x y p r o l i n e o b s e r v e d , n o r w o u l d i t e x p l a i n o b s e r v a t i o n s i n o u r l a b o r a t o r y that g l y c i n e and t y r o s i n e a r e i n c o r p o r a t e d at r a t e s s i m i l a r to that of i n c o r p o r a t i o n of p r o l i n e into the c o l l a g e n of g r a n u l o m a obtained f r o m s c o r b u t i c a n i m a l s . The s t r i k i n g i n c r e a s e s i n the f o r m a t i o n of t r i t i a t e d w a t e r and t r i tiated h y d r o x y p r o l i n e on i n v i t r o a d d i t i o n of a s c o r b a t e a r e consistent w i t h a function of this v i t a m i n i n hydroxylation—probably at step 3. The p r e s e n t r e s u l t s do not support a s y s t e m i c a s c o r b i c a c i d - m e d i a t e d ef fect, the b e l i e f that a s c o r b i c a c i d functions i n the maintenance of c o l lagen, o r a c t s by s t i m u l a t i n g m a t u r a t i o n of the f i b r o b l a s t s i n the s y s t e m under study h e r e . T h e p r e s e n t data do not support the p o s s i b i l i t y that i n t e r m e d i a t e s containing h y d r o x y p r o l i n e a c c u m u l a t e i n s c u r v y . The p r o p o s a l that a s c o r b i c a c i d i s i n v o l v e d i n the h y d r o x y l a t i o n r e a c t i o n i t s e l f i s consistent w i t h s t u d i e s on the nonenzymatic h y d r o x y l a t i o n of p r o l i n e (4) and on e n z y m a t i c h y d r o x y l a t i o n of other compounds (5, 20, 2 1 , 44, 51). A m o r e e l a b o r a t e and somewhat s p e c u l a t i v e s c h e m e for the f o r m a t i o n of c o l l a g e n and the c o n v e r s i o n of p r o l i n e to c o l l a g e n h y d r o x y p r o line i s : , E - p r o -A M Ρ Proline' ^^[pro]

-pr ο- sRNA ^^collagen

"-[hypro]—•hypro-sRNA"''^ hydroxyproline

A c c o r d i n g to this p r o p o s a l , the i n c o r p o r a t i o n of p r o l i n e into c o l l a gen f o l l o w s the a c y l adenylate and a c y l R N A stages now g e n e r a l l y b e l i e v e d to o c c u r i n p r o t e i n s y n t h e s i s . A bound h y d r o x y p r o l i n e i n t e r m e diate i s postulated, f r o m w h i c h h y d r o x y p r o l i n e i s t r a n s f e r r e d to s o l u b l e R N A . We p r e f e r to suggest that different R N A a c c e p t o r m o l e c u l e s e x i s t f o r h y d r o x y p r o l i n e and p r o l i n e ; t h i s w o u l d be consistent w i t h r e c e n t w o r k w h i c h i n d i c a t e s that the s o l u b l e R N A m o l e c u l e contains the i n f o r m a t i o n f o r i n c o r p o r a t i o n of a p a r t i c u l a r a m i n o a c i d into p r o t e i n (3). A l t h o u g h i t i s c o n c e i v a b l e that t h e r e i s h y d r o x y l a t i o n of p r o l y l - s R N A to y i e l d h y d r o x y p r o l y l - s R N A , an a d d i t i o n a l m e c h a n i s m w o u l d be needed



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to d i s t i n g u i s h the two types of a c y l r i b o n u c l e i c a c i d s . If the template r e c o g n i z e s only the s o l u b l e R N A and not the a m i n o a c i d , h y d r o x y l a t i o n m u s t o c c u r p r i o r to a c y l - R N A f o r m a t i o n . If two types of p r o l y l - R N A e x i s t (one of the p r e c u r s o r of c o l l a g e n p r o l i n e and the other that of c o l l a g e n h y d r o x y p r o l i n e ) , the h y d r o x y l a t i o n s y s t e m as w e l l as the t e m plate m u s t be able to d i s t i n g u i s h between the two types of R N A m o l e c u l e s . A c c o r d i n g to the p r o p o s e d s c h e m e , one w o u l d p r e d i c t a s p e c i f i c nucleotide sequence code f o r h y d r o x y p r o l i n e (or the bound h y d r o x y p r o line intermediate). T h e r e i s evidence that f r e e h y d r o x y p r o l i n e c a n be i n c o r p o r a t e d into c o l l a g e n to s o m e extent. T h u s , M i t o m a (26) found that free h y d r o x y p r o l i n e w a s i n c o r p o r a t e d into c h i c k e m b r y o c o l l a g e n at about 10% of the r a t e o b s e r v e d w i t h p r o l i n e . T h e s c h e m e given above i n d i c a t e s a p a t h way by w h i c h t h e r e may be a r e l a t i v e l y s l o w influx of h y d r o x y p r o l i n e into the h y d r o x y p r o l i n e i n t e r m e d i a t e . S i m i l a r l y , the breakdown of this i n t e r m e d i a t e might be r e s p o n s i b l e f o r the f o r m a t i o n of free h y d r o x y p r o l i n e o b s e r v e d i n s o m e t i s s u e s and p o s s i b l y a l s o that found i n u r i n e . If t h i s s c h e m e i s c o r r e c t , i t should be p o s s i b l e to obtain conditions under w h i c h p r o t e i n s y n t h e s i s but not h y d r o x y l a t i o n i s b l o c k e d . That t h i s m a y be p o s s i b l e e x p e r i m e n t a l l y i s suggested by the r e s u l t s of r e cent e x p e r i m e n t s (24) i n our l a b o r a t o r y , w h i c h have shown that i n the p r e s e n c e of p u r o m y c i n , the f o r m a t i o n of c o l l a g e n h y d r o x y p r o l i n e d e t e r m i n e d i n c a r a g e e n i n t u m o r s as d e s c r i b e d h e r e i s m a r k e d l y reduced, but that h y d r o x y l a t i o n (as d e t e r m i n e d by the f o r m a t i o n of t r i t i a t e d w a t e r and t r i t i a t e d h y d r o x y p r o l i n e ) continues. F u r t h e r w o r k — e s p e c i a l l y eff o r t s d i r e c t e d at i s o l a t i o n of i n t e r m e d i a t e s — a r e i n p r o g r e s s . M a n n e r and G o u l d (22) have r e p o r t e d that the f o r m a t i o n of free h y d r o x y p r o l i n e i n c h i c k e m b r y o s t r e a t e d w i t h p u r o m y c i n continues i n the absence of s i g n i f i c a n t c o l l a g e n f o r m a t i o n . T h i s finding i s analogous to o u r o b s e r v a t i o n s on c a r r a g e e n i n t u m o r s . M a n n e r and G o u l d a l s o o b tained evidence f o r the f o r m a t i o n of h y d r o x y p r o l y l - R N A i n c h i c k e m b r y o s , and concluded that p r o l i n e can be c o n v e r t e d to h y d r o x y p r o l i n e without p r i o r i n c o r p o r a t i o n of p r o l i n e into p r o t e i n . O n the other hand, P e t e r k o f s k y and Udenfriend concluded i n a p r e l i m i n a r y r e p o r t (30) that a m i c r o s o m a l R N A - b o u n d polypeptide of c o n s i d e r a b l e s i z e i s the s u b s t r a t e f o r h y d r o x y l a t i o n . The l a t t e r c o n c l u s i o n was based on studies w i t h a c e l l - f r e e c h i c k e m b r y o s y s t e m i n w h i c h p u r o m y c i n and r i b o n u c l e a s e w e r e added at v a r i o u s t i m e i n t e r v a l s d u r i n g the c o u r s e of i n c u bation w i t h l a b e l e d p r o l i n e . M a n n e r and G o u l d (22) and P e t e r k o f s k y and Udenfriend (30) o b s e r v e d l a g p e r i o d s i n the f o r m a t i o n of h y d r o x y p r o l i n e (compare F i g u r e s 2 and 3). A d d i t i o n a l study on the nature of the i n t e r m e d i a t e s i n v o l v e d i n the i n c o r p o r a t i o n of p r o l i n e into the c o l l a g e n i m i n o a c i d s i s needed. Conclusions T h e p r e s e n t studies have shown that the i n v i t r o c a r r a g e e n i n t u m o r s y s t e m can be employed w i t h t r i t i a t e d p r o l i n e to p r o v i d e a p r o c e d u r e s a t i s f a c t o r y f o r f o l l o w i n g h y d r o x y l a t i o n by m e a s u r e m e n t of t r i t i a t e d w a t e r . T h e data support a dual-pathway m e c h a n i s m of p r o l i n e i n c o r -



80

p o r a t i o n and i n d i c a t e that a s c o r b i c a c i d functions at the h y d r o x y l a t i o n step. T h e s e studies suggest s p e c i f i c e x p e r i m e n t s w i t h c e l l - f r e e s y s t e m s i n w h i c h i n t e r m e d i a t e s containing p r o l i n e and h y d r o x y p r o l i n e , and subsequently, s p e c i f i c e n z y m e s , m a y be i s o l a t e d .


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